TRAF Inhibitors

ABSTRACT

The invention concerns novel inhibitors of tumor necrosis factor receptor associated factor(TRAF) mediated signal transduction. The invention encompasses the novel inhibitor proteins (I-TRAFs), nucleic acid encoding them, methods for their recombinant production, and their use in screening assays and as pharmaceuticals.

This is a divisional of non-provisional application Ser. No. 08/700,749filed Aug. 14, 1996, now U.S. Pat. No. 5,789,550 claiming priority underSection 119(e) to provisional Application Ser. No. 60/002382 filedAugust 17, 1995, the entire disclosure of which is hereby incorporatedby reference.

FIELD OF THE INVENTION

The present invention concerns the isolation, recombinant production andcharacterization of novel polypeptides which block signal transductionmediated by tumor necrosis factor associated factors (TRAFs), includingTRAF2. In particular, this invention concerns novel proteins that act asspecific inhibitors of TRAF2-dependentNF-κB activation signaled bycertain members of the TNF receptor superfamily, such as TNF-R2 andCD40, methods and means for making them and their use in screeningassays or as pharmaceuticals.

BACKGROUND OF THE INVENTION

Tumor necrosis factor (TNF) is a cytokine produced mainly by activatedmacrophages which elicits a wide range of biological effects. Theseinclude an important role in endotoxic shock and in inflammatory,immunoregulatory, proliferative, cytotoxic, and anti-viral activities(reviewed by Goeddel, D. V. et al., Cold Spring Harbor Symposia onQuantitative Biology 51, 597-609 [1986]; Beutler, B. and Cerami, A.,Ann. Rev. Biochem. 57, 505-518 [1988]; Old, L. J., Sci. Am. 258(5),59-75 [1988]; Fiers, W. FEBS Lett. 285(2), 199-212 [1991]). Theinduction of the various cellular responses mediated by TNF is initiatedby its interaction with two distinct cell surface receptors, anapproximately 55 kDa receptor termed TNF-R1 and an approximately75 kDareceptor termed TNF-R2. Human and mouse cDNAs corresponding to bothreceptor types have been isolated and characterized (Loetscher, H. etal., Cell 61, 351 [1990]; Schall, T. J. et al., Cell 61, 361 [1990];Smith, C. A. et al., Science 248, 1019 [1990]; Lewis, M. et al., Proc.Natl. Acad. Sci. USA 88, 2830-2834 [1991]; Goodwin, R. G. et al., Mol.Cell. Biol. 11, 3020-3026 [1991]. Both TNF-Rs share the typicalstructure of cell surface receptors including extracellular,transmembrane and intracellular regions. The extracellularportions ofboth receptors are found naturally also as soluble TNF-binding proteins(Nophar, Y. et al., EMBO J. 9, 3269 [1990] and Kohno, T. et al., Proc.Natl. Acad. Sci. U.S.A. 87, 8331 [1990]). The amino acid sequence ofhuman TNF-R1 and the underlying nucleotide sequence are disclosed in EP417,563 (published Mar. 20, 1991), whereas EP 418,014 (published Mar.20, 1991) discloses the amino acid and nucleotide sequences of humanTNF-R2.

Both TNF receptors are independently active in signaling TNF responses.Direct signaling by TNF-R2 has been observed in lymphoid cells in whichTNF-R2 stimulates the proliferation of thymocytes and a murine cytotoxicT cell line CT6 (Tartaglia et al., Proc. Natl. Acad. Sci. USA 88,9292-9296 [1991]; Tartaglia et al, J. Immunol. 151, 4637-4641 [1993]).Both TNF-R1 and TNF-R2, along with other members of the TNF receptorsuperfamily, e.g. CD40, have been shown to independently mediate theactivation of the transcription factor NF-κB (Lenardo & Baltimore, Cell58: 227-229 [1989]; L.ae butted.greid, A., et al., J. Biol. Chem. 269,7785-7791 [1994]; Rothe et al., Cell 78, 681-692 [1994]; Wiegmann etal., J. Biol. Chem. 267, 17997-18001 [1992]). NF-κB is a member of theRel family of transcriptional activators that control the expression ofa variety of important cellular and viral genes (Lenardo & Baltimore,supra, and Thanos and Maniatis, Cell 80, 529-532 [1995]). TNF-R2 alsomediates the transcriptional induction of the granulocyte-macrophagecolony stimulating factor (GM-CSF) gene (Miyatake et al., EMBO J. 4:2561-2568 [1985]; Stanley et al., EMBO J. 4: 2569-2573 [1985]) and theA20 zinc fingerprotein gene (Opipari et al., J. Biol. Chem. 265:14705-14708 [1990]) in CT6 cells, and participates as an accessorycomponent to TNF-R1 in the signaling of responses primarily mediated byTNF-R1, like cytotoxicity (Tartaglia, L. A. and Goeddel, D. V., Immunol.Today 13, 151-153 [1992]).

Recent research has lead to the isolation of polypeptide factorsassociated with the intracellulardomain of the 75 kDa tumor necrosisfactor receptor, TNF-R2 ("tumor necrosis factor receptor associatedfactors" or "TRAFs") which participate in the TNF-R2 signal transductioncascade. TRAF1 and TRAF2 were the first two identified members of thisnovel protein family containing a novel C-terminal homology region, theTRAF domain (Rothe et al., Cell 78, 681-692 [1994]; U.S. Pat. Nos.5,670,319 issued on Sep. 23, 1997, and 5,708,142 issued Jan. 13, 1998. Afurther TRAF domain protein, TRAF3 (originally termed CD40bp, CRAF, orLAP1) has also been identified (Hu et al., J. Biol. Chem. 269, 30069[1994]; Cheng et al., Science 267, 1494 [1995], and Mosialos et al.,Cell 80, 389 [1995]). TRAFs transduce signals from TNF-R2, CD40 andpresumably from other members of the TNF receptor superfamily that alsoincludes the low affinity nerve growth factor receptor, the Fas antigen,CD27, CD30, OX40, 4-1BB, and TNFR-RP (Rothe et al., supra; Hu et al., J.Biol. Chem. 269, 30069-30072 [1994]; Cheng et al., Science 267,1494-1498 [1995]; Mosialos et al., Cell 80, 389-399 [1995]; Rothe etal., Science, in press; Smith et al, Cell 76, 959-962 [1994]). Inaddition to the shared conserved C-terminal TRAF domain that is involvedin both receptor association and oligomerization, TRAF2 and TRAF3 eachcontain an N-terminal RING finger domain and five zinc finger structuresof weak sequence similarity. CD40 and TNF-R2 interact directly withTRAF2 and indirectly with TRAF1 via a TRAF2/TRAF1 heterodimer. TRAF2 (orthe TRAF2/TRAF1 heterodimer) is required for CD40- and TNF-R2-mediatedactivation of the transcription factor NF-κB. TRAF3 interacts with CD40and self-associates,but does appear to not associate with TNF-R2, TRAF1,or TRAF2. The role of TRAF3 in signal transduction is less well defined,but it may antagonize the effects of TRAF2 (Rothe et al., Science, inpress). The TRAF proteins also interact with the C-terminal cytoplasmicdomain of the Epstein-Barr virus transforming protein LMP1 (Mosialos etal, Cell 80, 389 [1995]). LMP1 is a dominant oncogene that has multipledownstream effects on cell growth and gene expression, at least some ofwhich require NF-κB activation (Laherty et al., J. Biol. Chem. 267,24157 [1992]; Rowe et al., J. Virol. 68, 5602 [1994]). TRAF2 is believedto be a common signal transducer for TNF-R2, CD40 and LMP1.

SUMMARY OF THE INVENTION

The present invention is based on the identification, recombinantproduction and characterization of certain novelTRAF-interactingproteins named "inhibitors of TRAF" (I-TRAFs). Morespecifically, the present invention is based on the isolation of cDNAsencoding various forms of murine and human I-TRAFs encoding proteinswith no significant sequence similarity to any known protein, and on theexpression and characterization of the encoded I-TRAF proteins.

In one aspect, the invention concerns purified I-TRAF proteins whichinhibit the interaction of TRAFs with members of the TNF receptorsuperfamily, such as TNF-R2, CD40, and/or other cellular proteins withwhich TRAF proteins are normally, directly or indirectly, associated,such as LMP1.

In particular, the invention concerns an isolated I-TRAF polypeptidecomprising the amino acid sequence of a polypeptide selected from thegroup consisting of:

(a) a native sequence I-TRAF polypeptide,

(b) a polypeptide having at least about 70% amino acid sequence identitywith the TRAF-binding domain of a native sequence I-TRAF polypeptide andcapable of inhibiting a TRAF-mediated signaling event, and

(c) a fragment of a polypeptide of (a) or (b) capable of inhibiting aTRAF-mediated signaling event.

Preferably, the I-TRAF polypeptides of the present invention have an atleast about 60% overall amino acid sequence identity with a nativesequence I-TRAF polypeptide. The I-TRAF polypeptides preferably comprisea fragment of a native mammalian I-TRAF protein that is necessary andsufficient for association with a native TRAF protein. More preferably,the I-TRAF polypeptides comprise a fragment of a native mammalian I-TRAFor a polypeptide sufficiently homologous to retain the ability ofinteractingwith the TRAF domain of a native mammalian TRAF protein. Evenmore preferably, the I-TRAF polypeptides comprise the N-terminal portionof a native mammalian I-TRAF capable of interaction with a native TRAF,preferably TRAF2. The mammalian proteins are preferably human.

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding I-TRAF proteins which inhibit the interaction of TRAFswith members of the FNF receptor superfamily, such as TNF-R2, CD40,and/or other intracellular proteins with which TRAF proteins arenormally, directly or indirectly, associated, such as LMPI. The nucleicacid preferably comprises a nucleotide sequence encoding a 413 or 447amino acid murine I-TRAF protein (SEQ. ID. NOs: 1 and 3) or a 425 aminoacid human I-TRAF protein (SEQ. ID. NO: 5) or a functional derivativethereof, including the truncated murine protein shown in FIG. 1 (SEQ.ID. NO: 8). In another preferred embodiment, the nucleic acid is capableof hybridizing to the complement of any of the nucleic acid moleculesrepresented by SEQ. ID. NOs: 2, 4 and 6.

The invention further concerns vectors, cells and organisms comprisingsuch nucleic acid.

In a further aspect, the invention concerns a screening assay foridentifying molecules that modulate the I-TRAF/TRAF binding. Preferably,the molecules either prevent I-TRAF/TRAF interaction or prevent orinhibit dissociation of I-TRAF/TRAF complexes. The assay comprises theincubation of a mixture comprising TRAF and I-TRAF with a candidatemolecule and detection of the ability of the candidate molecule tomodulate I-TRAF/TRAF binding, e.g. to prevent the interaction of I-TRAFwith TRAF or to prevent or inhibit the dissociation of I-TRAF/TRAFcomplexes. The screened molecules preferably are small molecule drugcandidates.

In another aspect, the invention relates to an assay for identifying amolecule the signal transduction of which is mediated by the associationof a TRAF with a cellular protein, comprising (a) incubating a mixturecomprising a TRAF, and an I-TRAF with a candidate molecule, and (b)detecting the ability of the candidate molecule to release TRAF from thecomplex formed with I-TRAF a TRAF-mediated signaling event.

In yet another aspect, the invention concerns a method of treating atumor associated with Epstein-Barrvirus comprising administering to apatient having developed or at risk of developing such tumor atherapeutically effective amount of an I-TRAF. Pharmaceuticalcompositions comprising an I-TRAF as an active ingredient are alsowithin the scope of the present invention.

The invention also concerns a method for modulating TRAF-mediated signaltransduction in a cell comprising introducing into the cell a nucleicacid encoding an I-TRAF or a functional derivative.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1(a) shows the deduced amino acid sequences of murine and humanI-TRAFs. The translationinitiation codons of the murine I-TRAFα and -βsplice variants are indicated by α and β, respectively. Thesplicejunction is marked by an asterisk (*). All of the isolated humanI-TRAF cDNA clones correspond to murine I-TRAFα. The initiatormethionine of human I-TRAF is preceded by an upstream in-frame stopcodon not present in the murine I-TRAFα cDNA clones. The amino acidsequences of two additional splice variants of mouse I-TRAFα, ending inpremature termination codons, are also listed.

FIG. 1(b) shows the results of Northern blot analysis of I-TRAF mRNA inmultiple mouse tissues.

FIG. 2 shows that I-TRAF associates with TRAF1, TRAF2 and TRAF3 in an invitro binding assay.

FIG. 3 shows the results of Western blot analysis which demonstrate thatI-TRAF blocks the association of TRAF2 with TNF-R2.

FIG. 4 illustrates the inhibitory effect of I-TRAF on activation ofNF-κB.

SEQ. ID. NO: 1 shows the amino acid sequence of the 413 aa murine I-TRAF(murine I-TRAFα), starting with the amino acid marked "α" in FIG. 1.

SEQ. ID. NO: 2 shows the nucleotide sequence of murine I-TRAFα. Thissequence was obtained from a composite of several independent clonedsequenced.

SEQ. ID. NO: 3 shows the additional 34 amino acids present in thesequence of the 447 aa murine I-TRAF (murine I-TRAFβ), starting with theamino acid marked "β" in FIG. 1.

SEQ. ID. NO: 4 shows the nucleotide sequence encoding the additional 34amino acids of murine I-TRAFβ.

SEQ. ID. NO: 5 shows the amino acid sequence of full-length humanI-TRAF.

SEQ. ID. NO: 6 shows the nucleotide sequence of full-length humanI-TRAF.

SEQ. ID. NO: 7 is the complete, unedited nucleotide sequence of one fulllength clone encoding murine I-TRAFα (clone 8).

SEQ. ID. NO: 8 is the nucleotide sequence encoding the truncated murineI-TRAFα protein ending by the sequence VTVLH shown in FIG. 1 (clone 23).

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

The phrases "inhibitor of TRAF", "inhibitor of TRAF protein", "inhibitorof TRAF polypeptide", "I-TRAF protein", "I-TRAF polypeptide" and"I-TRAF" are used interchangeably and refer to a native sequencepolypeptide which inhibits the interaction of a native TRAF with a cellsurface receptor that triggers TRAF-mediated NF-κB activation cascades,such as a member of the TNF receptor superfamily (e.g. TNF-R2, CD40and/or CD30) and/or with another cellular protein with which the nativeTRAF is normally (directly or indirectly) associated, provided that suchI-TRAF polypeptide is other than a blocking antibody, and functionalderivatives thereof. The I-TRAF protein preferably exerts its inhibitoryfunction by binding to a TRAF protein, such as TRAF1, TRAF2 and/orTRAF3, keeping it in a latent state from which the TRAF protein can bereleased, for example, by ligand-induced receptor aggregation. Thephrases "native sequence polypeptide" and "native polypeptide" and theirgrammatical variants are used interchangeably and designate apolypeptide as occurring in nature in any cell type of any human ornon-human animal species, with or without the initiating methionine,whether purified from native source, synthesized, produced byrecombinant DNA technology or by any combination of these and/or othermethods. Native sequence I-TRAF polypeptides specifically include the413 amino acid murine I-TRAF (I-TRAFα; SEQ. ID. NO: 1), the 447 aminoacid murine I-TRAF (I-TRAFβ; SEQ. ID. NO: 3), and the 425 amino acidhuman I-TRAF (SEQ. ID. NO: 5), and their additional, naturally occurringalternative splice and allelic variants. Native I-TRAF polypeptides fromother mammalian species, such as porcine, canine, equine, etc. are alsoincluded.

The phrases "TRAF", "TRAF protein" and "TRAF polypeptide" are usedinterchangeably and refer to a native sequence factor capable ofspecific association with the intracellular domain of a native member ofthe TNF receptor superfamily (such as TNF-R2, CD40 or CD30), andfunctional derivatives of such native factor. In the context of thisdefinition, the phrase "specific association" is used in the broadestsense, and includes direct binding to a site or region within theintracellular domain of a native TNF receptor superfamily member of thehuman or of any other animal species, and indirect association mediatedby a further molecules, such as another TRAF. The phrase "nativesequence factor" is defined in an analogous manner to the definition of"native sequence polypeptide" provided above. The native sequence TRAFpolypeptides specifically include the native murine TRAF1 and TRAF2polypeptides disclosed in Rothe et al., Cell 78, 681-692 [1994]), TRAF3as disclosed in Hu et al., supra; Cheng el al., supra; and Mosialos etal., supra, and their equivalents in human and other animal species.

A "functional derivative" of a native polypeptide is a compound having aqualitative biological activity in common with the native polypeptide.For the purpose of the present invention, a "functional derivative" of anative I-TRAF is defined by its ability to inhibit the association of aTRAF with a member of the TNF receptor superfamily with which the TRAFis naturally associated, with the proviso that such functionalderivatives are not (anti-TRAF or anti-TNF receptor superfamily member)blocking antibodies. Preferably, a functional derivative binds to anative TRAF polypeptide, such as TRAF1, TRAF2 and/or TRAF3 thereby(reversibly or irreversibly) preventing its interaction with cellsurface receptors (e.g., TNF-R2, CD40, CD30) that trigger TRAF-mediatedNF-κB activation cascades. In a particularly preferred embodiment, theprevention of TRAF-mediated signaling is reversible, and the TRAFs canbe released from their latent, inhibited state, e.g. by ligand-mediatedreceptor aggregation. The functional derivatives preferably have atleast about 60%, more preferably at least about 70%, even morepreferably at least about 80%, most preferably at least about 90%overall amino acid sequence identity with a native sequence I-TRAFpolypeptide, preferably a human I-TRAF. Even more preferably, thefunctional derivatives show at least about 70%, more preferably at leastabout 80% and most preferably at least about 90% amino acid sequenceidentity with the TRAF-binding domain of a native sequence I-TRAFpolypeptide. Fragments of native sequence I-TRAF polypeptides fromvarious mammalian species and sequences homologous to such fragmentsconstitute another preferred group of I-TRAF functional derivatives.Such fragments preferably include the N-terminal region of a nativesequence I-TRAF which is necessary and sufficient for TRAF binding, suchas amino acids 35-236 of the mouse I-TRAF sequences represented in SEQ.ID. NO: 1 or 3, and the equivalent part of a human I-TRAF sequence, orshow at least about 70%, more preferably at least about 80%, mostpreferably at least about 90% sequence identity with the TRAF-bindingN-terninal portion of a native sequence I-TRAF. Another preferred groupof I-TRAF functional derivatives is encoded by nucleic acid hybridizingunder stringent conditions to the complement of nucleic acid encoding anative I-TRAF polypeptide.

Functional derivatives of native TRAF polypeptides, as defined for thepurpose of the present invention, are characterized by retaining theability of native TRAFs to associate (directly or indirectly) with theintracellulardomain of a member of the TNF receptor superfamily, such asTNF-R2, CD40 and/or CD30. Such TRAF functional derivatives preferablyretain or mimic the region(s) within a native TRAF sequence thatdirectly participate(s) in association with the intracellular domain ofa TNF receptor superfamily member and/or in homo- or heterodimerization,along with the region(s) to which a native I-TRAF polypeptide binds. Apreferred group of TRAF functional derivatives shows at least about 60%,more preferably at least about 70%, even more preferably at least about80%, most preferably at least about 90% overall sequence identity with anative sequence TRAF. Other preferred functional derivatives are orcomprise the conserved TRAF domains of native sequence TRAF proteins.Within this group, TRAF fragments comprising at least amino acids 264 to501 of the native TRAF2 amino acid sequence are particularly preferred.Also preferred are TRAF variants which show at least about 70%, morepreferably at least about 80%, most preferably at least about 80% aminoacid sequence identity with the TRAF region of a native sequence TRAFprotein, preferably TRAF2. A further preferred group of TRAF functionalderivatives is encoded by nucleic acid capable of hybridizing understringent conditions to the complement of a native sequence TRAFpolypeptide of any mammalian species, including humans.

"Identity" or "homology" with respect to a native polypeptide orpolypeptide and its functional derivative herein is the percentage ofamino acid residues in the candidate sequence that are identical withthe residues of a corresponding native polypeptide, after aligning thesequences and introducing gaps, if necessary, to achieved the maximumpercent homology, and not considering any conservative substitutions aspart of the sequence identity. Methods and computer programs for thealignment are well known in the art,

"Stringent conditions" can be provided in a variety of ways, such as byovernight incubation at 42° C. in a solution comprising: 20% formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA. Alternatively, the stringentconditions are characterized by a hybridization buffer comprising 30%formamide in 5×SSPE (0.18 M NaCl, 0.01 M NaPO₄, pH 7.7, 0.0001 M EDTA)buffer at a temperature of 42° C., and subsequent washing at 42° C. with0.2×SSPE. Preferably, stringent conditions involve the use of ahybridization buffer comprising 50% formamide in 5×SSPE at a temperatureof 42° C. and washing at the same temperature with 0.2×SSPE.

An "isolated" polypeptideor nucleic acid is unaccompaniedwith at leastsome of the material with which it is associated in its nativeenvironment. An isolated polypeptide constitutes at least about 2% byweight, and preferably at least about 5% by weight of the total proteinin a given sample. An isolated nucleic acid constitutes at least about0.5% by weight, and preferably at least about 5% by weight of the totalnucleic acid present in a given sample.

The term "antibody" is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist and antagonistantibodies), antibody compositions with polyepitopic specificity, aswell as antibody fragments (e.g., Fab, F(ab')₂, and Fv), so long as theyexhibit the desired biological activity.

The term "monoclonal antibody" as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier "monoclonal"indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler & Milstein, Nature 256:495 (1975), or may be made byrecombinant DNA methods [see, e.g. U.S. Pat. No. 4,816,567 (Cabilly etal.)].

The monoclonal antibodies herein specifically include "chimeric"antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity [U.S. Pat. No. 4,816,567;Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA 81,6851-6855 (1984)].

"Humanized" forms of non-human (e.g. murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibody maycomprise residues which are found neither in the recipient antibody norin the imported CDR or framework sequences. These modifications are madeto further refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulinand allor substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulinconstant region(Fc), typically that of a human immunoglobulin. For further details see:Jones et al., Nature 321, 522-525 (1986); Reichmann et al., Nature 332,323-329 (1988); Presta, Curr. Op. Struct. Biol. 2 593-596 (1992) andU.S. Pat. No. 5,225,539 (Winter) issued Jul. 6, 1993.

A "vector" as defined for the purpose of the present invention refers toa DNA construct containing a DNA sequence which is operably linked to asuitable control sequence capable of effecting the expression of the DNAin a suitable host. Such control sequences include a promoter to effecttranscription, an optional operator sequence to control suchtranscription, a sequence encoding suitable mRNA ribosome binding sites,and sequences which control the termination of transcription andtranslation. The vector may be a plasmid, a phage particle, or simply apotential genomic insert. Once transformed into a suitable host, thevector may replicate and function independently of the host genome, ormay, in some instances, integrate into the genome itself. In the presentspecification "plasmid" and "vector" are sometimes used interchangeablyas the plasmid is the most commonly used form of vector at present.However, the invention is intended to include such other forms ofvectors which serve equivalent functions and which are, or become, knownin the art. Preferred expression vectors for mammalian cell cultureexpression are based on pRK5 (EP 307,247; Rothe et al., Cell, supra) andpSVI6B (PCT Publication No WO 91/08291).

In the context of the present invention the expressions "cell", "cellline", and "cell culture" are used interchangeably, and all suchdesignations include progeny. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological property, as screened for in the originally transformed cell,are included.

The "host cells" used in the present invention generally are prokaryoticor eukaryotic hosts. Such host cells are, for example, disclosed in U.S.Pat. No. 5,108,901 issued Apr. 28, 1992, and in copending applicationSer. No. 08/446,915 filed May 22, 1995 and its parent applications.Suitable prokaryotes include gram negative or gram positive organisms,for example E. coli or bacilli. A preferred cloning host is E. coli 294(ATCC 31,446) although other gram negative or gram positive prokaryotessuch as E. coli B, E. coli×1776 (ATCC 31,537), E. coli W3110 (ATCC27,325), Pseudomonas species, or Serratia Marcesans are suitable. Inaddition to prokaryotes, eukaryotic microbes such as filamentous fungiand yeasts are suitable hosts for appropriate vectors of the invention.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species and strains are commonly available and usefulherein, such as those disclosed in the above-cited patent and patentapplications. A preferred yeast strain for the present invention isSaccharoinyces cerevisiae HF7c (CLONTECH).

Suitable host cells may also derive from multicellularorganisms. Suchhost cells are capable of complex processing and glycosylationactivities. In principle, any higher eukaryotic cell culture isworkable, whether from vertebrate or invertebrate culture, althoughcells from mammals such as humans are preferred. Examples ofinvertebrate cells include plant and insect cells, see, e.g. Luckow etal., Bio/Technology 6, 47-55 (1988); Miller et al. in: GeneticEngineering, Setlow et al., eds., Vol. 8 (Plenum Publishing, 1986), pp.277-279; and Maeda et al., Nature 315, 592-594 (1985). Interest has beengreatest in vertebrate cells, and propagation of vertebrate cells inculture (tissue culture) is per se known. See, Tissue Culture, AcademicPress, Kruse and Patterson, eds. (1973). Examples of useful mammalianhost cell lines are monkey kidney CV1 line transformed by SV40 (COS-7,ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen.Virol. 36, 59 [1977]); baby hamster kidney cells 9BHK, (ATCC CCL 10);Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl.Acad. Sci. USA 77, 4216 [1980]); mouse sertolli cells (TM4, Mather,Biol. Reprod. 23, 243-251 [1980]); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci. 383, 44068 [1982]); MRC 5 cells; FS4 cells; and ahuman hepatoma cell line (Hep G2). Preferred host cells are humanembryonic kidney 293 and Chinese hamster ovary cells. Particularlypreferred for the present invention are the murineinterleukin-2-dependentcytotoxic T cell line CT6, and the humanembryonic kidney cell line 293, which are maintained as described inTartaglia et al., Proc. Natl. Acad. Sci. USA 88, 9292-9296 (1991) andPennica et al., J. Biol. Chem. 267, 21172-21178 (1992).

"Dissociation" is the process by which two molecules cease to interact:the process occurs at a fixed average rate under specific physicalconditions.

B. Identification and Purification of Native I-TRAFs

The native I-TRAF polypeptides may, for example, be identified andpurified from certain tissues which possess a TRAF (e.g. TRAF2) mRNA andto express it at a detectable level. Murine I-TRAF can, for example, beobtained from the murine fetal liver stromal cell line 7-4 (FL; Rothe etal., Cell supra) or from murine peripheral lymph nodes (PLN; U.S. Pat.No. 5,304,640 issued Apr. 19, 1994). Human I-TRAF can, for example, bepurified from human peripheral lymph nodes. I-TRAF mRNA is also presentand expressed in brain, spleen, lung, liver, skeletal muscle, kidney andtestis tissues (see FIG. 1b), along with TRAF, e.g. TRAF2 mRNAs. NativeI-TRAFs can, for example, be identified and purified from tissuesexpressing their mRNAs based upon their ability to bind TRAF2 (oranother TRAF, e.g. TRAF1). Native I-TRAFs will coprecipitated withimmunoprecipitated TRAF2. In a preferred embodiment, radiolabeled TRAF2or a derivative is immunoprecipitated with protein A-agarose (OncogeneScience) or with protein A-Sepharose (Pharmacia). The immunoprecipitateis then analyzed by autoradiography or fluorography, depending on theradiolabel used. The I-TRAF proteins will coprecipitate with the TRAF2or its derivative, and can be further purified by methods known in theart, such as purification on an affinity column. For large-scalepurification a scheme similar to that described by Smith and Johnson,Gene 67, 31-40 (1988) can be used. A cell lysate containing theI-TRAF(s) to be purified is applied to a glutathione-S-transferase(GST)-TRAF fusion protein affinity column. Protein(s) bound to thecolumn is/are eluted, precipitated and isolated by SDS-PAGE underreducing conditions, and visualized, e.g. by silver staining. GST genefusion vectors (pGEX vectors) as well as kits for cloning and expressionof GST fusion systems are commercially available from Pharmacia (seePharmacia Catalog, 1994, pages 133; and 142-143).

C. Recombinant Production of I-TRAF Polypeptides

Preferably, the I-TRAF polypeptides are prepared by standard recombinantprocedures by culturing cells transfected to express I-TRAF polypeptidenucleic acid (typically by transforming the cells with an expressionvector) and recovering the polypeptide from the cells. However, it isenvisioned that the I-TRAF polypeptides may be produced by homologousrecombination, or by recombinant production methods utilizing controlelements introduced into cells already containing DNA encoding an TRAFpolypeptide. For example, a powerful promoter/enhancer element, asuppressor, or an exogenous transcription modulatory element may beinserted in the genome of the intended host cell in proximity andorientation sufficient to influence the transcription of DNA encodingthe desired I-TRAF polypeptide. The control element does not encode theI-TRAF polypeptide, rather the DNA is indigenous to the host cellgenome. One next screens for cells making the polypeptide of thisinvention, or for increased or decreased levels of expression, asdesired. General techniques of recombinant DNA technology are, forexample, disclosed in Sambrook et al., Molecular Cloning: A laboratoryManual, Second Edition (Cold Spring Harbor, New York: Cold Spring HarborLaboratory Press) 1989.

1. Isolation of DNA encoding the I-TRAF polypeptides

For the purpose of the present invention, DNA encoding a I-TRAFpolypeptide may, for example, be obtained from cDNA libraries preparedfrom tissue believed to possess a TRAF mRNA and to express it at adetectable level. For example, cDNA library can be constructed byobtaining polyadenylated mRNA from a cell line known to express a TRAFprotein, and using the mRNA as a template to synthesize double strandedcDNA. Human and non-human cell lines and tissues suitable for thispurpose have been listed hereinabove. Alternatively, DNA encoding newI-TRAF polypeptides can be obtained from cDNA libraries prepared fromtissue known to express a previously identified I-TRAF polypeptide at adetectable level. The I-TRAF polypeptide genes can also be obtained froma genomic library, such as a human genomic cosmid library.

Libraries, either cDNA or genomic, are screened with probes designed toidentify the gene of interest or the protein encoded by it. For cDNAexpression libraries, suitable probes include monoclonal and polyclonalantibodies that recognize and specifically bind to an I-TRAFpolypeptide. For cDNA libraries, suitable probes include carefullyselected oligonucleotide probes (usually of about 20-80 bases in length)that encode known or suspected portions of an I-TRAF polypeptide fromthe same or different species, and/or complementary or homologous cDNAsor fragments thereofthat encode the same or a similar gene. Appropriateprobes for screening genomic DNA libraries include, without limitation,oligonucleotides, cDNAs, or fragments thereof that encode the same or asimilar gene, and/or homologous genomic DNAs or fragments thereof.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures as described in Chapters 10-12 ofSambrook et al., Molecular Cloning: A Laboratory Manual, New York, ColdSpring Harbor Laboratory Press, (1989).

A preferred method of practicing this invention is to use carefullyselected oligonucleotide sequences to screen cDNA libraries from varioustissues. The oligonucleotide sequences selected as probes should besufficient in length and sufficiently unambiguous that false positivesare minimized. The actual nucleotide sequence(s) is/are usually designedbased on regions of an I-TRAF which have the least codon redundance. Theoligonucleotides may be degenerate at one or more positions. The use ofdegenerate oligonucleotides is of particular importance where a libraryis screened from a species in which preferential codon usage is notknown.

The oligonucleotide must be labeled such that it can be detected uponhybridization to DNA in the library being screened. The preferred methodof labeling is to use ATP (e.g., γ³² P) and polynucleotidekinase toradiolabel the 5' end of the oligonucleotide. However, other methods maybe used to label the oligonucleotide,including, but not limited to,biotinylation or enzyme labeling.

cDNAs encoding I-TRAFs can also be identified and isolated by otherknown techniques of recombinant DNA technology, such as by directexpression cloning or by using the polymerasechain reaction (PCR) asdescribed in U.S. Pat. No. 4,683,195, issued Jul. 28, 1987, in section14 of Sambrook et al., Molecular Cloning: A Laboratory Manual, secondedition, Cold Spring Harbor Laboratory Press. New York, 1989, or inChapter 15 of Current Protocols in Molecular Biology, Ausubel et al.eds., Greene Publishing Associates and Wiley-lnterscience 1991. Thismethod requires the use of oligonucleotide probes that will hybridize toDNA encoding an I-TRAF.

According to a preferred method for practicing the invention, the codingsequences for I-TRAF proteins can be identified in a recombinant cDNAlibrary or a genomic DNA library based upon their ability to interactwith TRAF proteins, e.g. TRAF2. For this purpose one can use the yeastgenetic system described by Fields and co-workers [Fields and Song,Nature (London) 340, 245-246 (1989); Chien et al, Proc. Natl. Acad. Sci.USA 88, 9578-9582 (1991)] as disclosed by Chevray and Nathans [Proc.Natl. Acad. Sci. USA 89, 5789-5793 (1992)]. Many transcriptionalactivators, such as yeast GAL4, consist of two physically discretemodular domains, one acting as the DNA-binding domain, while the otherone functioning as the transcription activation domain. The yeastexpression system described in the foregoing publications (generallyreferred to as the "two-hybrid system") takes advantage of thisproperty, and employs two hybrid proteins, one in which the targetprotein is fused to the DNA-binding domain of GAL4, and another, inwhich candidate activating proteins are fused to the activation domain.The expression of a GAL1-lacZ reporter gene under control of aGAL4-activated promoter depends on reconstitution of GAL4 activity viaprotein-protein interaction. Colonies containing interactingpolypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-proteininteractionsbetweentwo specific proteins using thetwo-hybridtechnique is commercially available from Clontech. This systemcan also be extended to map protein domains involved in specific proteininteractions as well as to pinpoint amino acid residues that are crucialfor these interactions. Yeast two-hybrid cloning can be performed asdescribed in Rothe et al., supra, using TRAF2 or its TRAF domain in theGAL4 DNA-binding domain vector pPC97 (Chevray and Nathans, supra) asbait.

Once the sequence is known, the gene encoding a particular I-TRAFpolypeptide can also be obtained by chemical synthesis, following one ofthe methods described in Engels and Uhlmann, Angew. Chem. Int. Ed. Engl.28, 716 (1989). These methods include triester, phosphite,phosphoramidite and H-phosphonate methods, PCR and other autoprimermethods, and oligonucleotide syntheses on solid supports.

2. Amino acid sequence variants of native I-TRAF proteins or fragments

Amino acid sequence variants of native I-TRAFs and I-TRAF fragments areprepared by methods known in the art by introducing appropriatenucleotide changes into a native or variant I-TRAF DNA, or by in vitrosynthesis of the desired polypeptide. There are two principle variablesin the constructions of amino acid sequence variants: the location ofthe mutation site and the nature of the mutation. With the exception ofnaturally-occurring alleles, which do not require the manipulation ofthe DNA sequence encoding the I-TRAF, the amino acid sequence variantsof I-TRAF polypeptides are preferably constructed by mutating the DNA,either to arrive at an allele or an amino acid sequence variant thatdoes not occur in nature. Methods for identifying target residues withinnative proteins and for making amino acid sequence variants are wellknown in the art, and are, for example, disclosed in U.S. Pat. No.5,108,901 issued Apr. 28, 1992, and in copending application Ser. No.08/446,915 filed May 22, 1995 and its parent applications. The preferredtechniques include alanine-scanning mutagenesis, PCR mutagenesis,cassette mutagenesis, the phageamid display method, details of which arealso found in general textbooks, such as, for example, Sambrook et al.,supra, and Current Protocols in Molecular Biology, Ausubel et al., eds.,supra.

A preferred group of the I-TRAF amino acid sequence variants of thepresent invention comprises the substitution, insertion and/or deletionof one or more amino acids in a region directly involved inTRAF-binding. Amino acid alterations within this region are expected toresult in genuine changes in the I-TRAF/TRAF binding affinity and mayyield variants with stronger or weaker binding affinity than nativeI-TRAFs, as desired.

Naturally-occurring amino acids are divided into groups based on commonside chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophobic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Conservative substitutions involve exchanging a member within one groupfor another member within the same group, whereas non-conservativesubstitutions will entail exchanging a member of one of these classesfor another. Variants obtained by non-conservativc substitutions areexpected to result in more significant changes in the biologicalproperties/function of the obtained variant.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Deletions are preferably introduced into regions notdirectly involved in the interaction with TRAF, although deletions,optionally in combination with other types of mutations, in theTRAF-binding domain are also contemplated.

Amino acid insertions include amino- and/or carboxyl-terminal fusionsranging in length from one residue to polypeptides containing a hundredor more residues, as well as intrasequence insertions of single ormultiple amino acid residues. Intrasequence insertions (i.e. insertionswithin the I-TRAF protein amino acid sequence) may range generally fromabout 1 to 10 residues, more preferably 1 to 5 residues, more preferably1 to 3 residues. Examples of terminal insertions include the I-TRAFpolypeptides with an N-terminal methionyl residue, an artifact of itsdirect expression in bacterial recombinant cell culture, and fusion of aheterologous N-terminal signal sequence to the N-terminus of the I-TRAFmolecule to facilitate the secretion of the mature I-TRAF fromrecombinant host cells. Such signal sequences will generally be obtainedfrom, and thus homologous to, the intended host cell species. Suitablesequences include STII or Ipp for E. coli, alpha factor for yeast, andviral signals such as herpes gD for mammalian cells.

Other insertional variants of the native I-TRAF molecules include thefusion of the N- or C-terminus of the I-TRAF molecule to immunogenicpolypeptides, e.g. bacterial polypeptides such as beta-lactamase or anenzyme encoded by the E. coli trp locus, or yeast protein, andC-terminal fusions with proteins having a long half-life such asimmunoglobulin regions (preferably immunoglobulin constant regions toyield immunoadhesins), albumin, or ferritin, as described in WO 89/02922published on Apr. 6, 1989. For the production of immunoglobulin fusionssee also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

Since it is often difficult to predict in advance the characteristics ofa variant TRAF, it will be appreciated that screening will be needed toselect the optimum variant. For this purpose biochemical or otherscreening assays, such as those described hereinbelow, will be readilyavailable.

Preferred amino acid sequence variants of native sequence I-TRAFpolypeptides have the sequences not required for TRAF-binding (e.g. mostof amino acids from position 237 to the C-terminus of the murine I-TRAFsequence) deleted, leaving the TRAF-interacting region as the solefunctionally intact domain. Such variants may additionallyhave aminoacid substitutions, insertions and/or deletions within theTRAF-interacting region, in order to optimize their binding properties,stability or other characteristics.

3. Insertion of DNA into a cloning/expression vehicle

Once the nucleic acid of a native or variant I-TRAF is available, it isgenerally ligated into a replicable expression vector for furthercloning (amplification of the DNA) or expression.

Expression and cloning vectors are well known in the art and contain anucleic acid sequence that enables the vector to replicate in one ormore selected host cells. The selection of an appropriate vector willdepend on 1) whether it is to be used for DNA amplification or for DNAexpression, 2) the size of the DNA to be inserted into the vector, and3) the host cell to be transformed with the vector. Each vector containsvarious components depending on its function (amplification of DNA orexpression of DNA) and the host cell for which it is compatible. Thevector components generally include, but are not limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Suitable expression vectors, for use incombination with a variety of host cells, are well known in the art, andtheir preferred representatives have been listed hereinabove.

The host cells are then transformed, cultured, and the geneamplification/expression is detected by methods well known in the art,such as those disclosed in Sambrook et al., supra, and Ausubel et al.,supra.

4. Isolation and purification of the I-TRAF polypeptides

The I-TRAF polypeptides are typically recovered from lysates ofrecombinant host cells. When I-TRAF is expressed in a recombinant hostcell other than one of human origin, it is completely free of proteinsor polypeptides of human origin. However, it is necessary to purify theI-TRAF protein from recombinant cell proteins or polypeptides to obtainpreparations that are substantially homogenous to the I-TRAF. As a firststep, the culture medium or lysate is centrifuged to remove particulatecell debris. The membrane and soluble fractions are then separated. TheI-TRAF protein may then be purified from the soluble protein fraction.The following procedures are exemplary of suitable purificationprocedures: fractionation or immunoaffinity on ion-exchange columns;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; and protein A Sepharose columns to remove contaminantsuch as IgG. The preferred scheme for purifying native I-TRAFs fromcells in which they naturally occur is equally suitable for thepurification of recombinant I-TRAFs, including functional derivatives ofthe native molecules, from recombinant host cells.

D. Covalent Modifications of I-TRAF Polypeptides

Covalent modificationsof I-TRAF are included within the scope herein.Such modifications are traditionally introduced by reacting targetedamino acid residues of the I-TRAF with an organic derivatizing agentthat is capable of reacting with selected sides or terminal residues, orby harnessing mechanisms of post-translationalmodificationsthat functionin selected recombinant host cells. The resultant covalent derivativesare useful in programs directed at identifying residues important forbiological activity, for immunoassays of the I-TRAF, or for thepreparation of anti-I-TRAF antibodies for immunoaffinity purification.For example, complete inactivation of the biological activity of theprotein after reaction with ninhydrin would suggest that at least onearginyl or lysyl residue is critical for its activity, whereafier theindividual residues which were modified under the conditions selectedare identified by isolation of a peptide fragment containing themodified amino acid residue. Such modifications are within the ordinaryskill in the art and are performed without undue experimentation.

Cysteinyl residues most commonly are reacted with a-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction withdiethylpyrocarbonateat pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1M sodium cacodylateat pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing a-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵ I or ¹³¹ I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides(R'--N═C═N--R') such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl, threonyl or tyrosylresidues, methylation of the α-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Propertics, W.H. Freeman & Co., San Francisco, pp. 79-86[1983]), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group. The molecules may further be covalentlylinked to nonproteinaceous polymers, e.g. polyethylene glycol,polypropylene glycol or polyoxyalkylenes, in the manner set forth inU.S. Ser. No. 07/275,296 or U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Derivatization with bifunctional agents is useful for preparingintramolecular aggregates of I-TRAFs with polypeptidesas well as forcross-linking the I-TRAF polypeptideto a water insoluble support matrixor surface for use in assays or affinity purification. In addition, astudy of interchain cross-links will provide direct information onconformational structure. Commonly used cross-linking agents include1,1-bis(diazoacetyl)-2-phenylethaneglutaraldehyde, N-hydroxysuccinimideesters, homobifunctional imidoesters, and bifunctional maleimides.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates which are capable of forming cross-links in the presenceof light. Alternatively, reactive water insoluble matrices such ascyanogen bromide activated carbohydrates and the systems reactivesubstrates described in U.S. Pat. Nos. 3,959,642; 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employedfor protein immobilization and cross-linking.

Certain post-translationalmodifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andaspariginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl, threonyl ortyrosyl residues, methylation of the α-amino groups of lysine, arginine,and histidine side chains [T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)].

The TRAF may be entrapped in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, in colloidaldrug delivery systems (e.g. liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th Edition, Osol, A., Ed. (1980).

E. Use of I-TRAF Polypeptides and Nucleic Acid Encoding Them

The nucleic acids encoding the I-TRAF polypeptides of the presentinvention find a variety of applications on their own, including use astranslatable transcripts, hybridization probes for identifying nucleicacid encoding I-TRAFs from other species and/or structurally relatedpolypeptides, PCR primers, and therapeutic nucleic acids, including genetherapy applications. In gene therapy applications, genes are introducedinto cells in order to achieve in vivo synthesis of atherapeuticallyeffectivegenetic product, for example for replacement ofa defective gene. "Gene therapy" includes both conventional gene therapywhere a lasting effect is achieved by a single treatment, and theadministrationof gene therapeutic agents, which involves the one time orrepeated administration of a therapeutically effective DNA or mRNA.Antisense RNAs and DNAs can be used as therapeutic agents for blockingthe expression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellularconcentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83, 4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducingnucleic acidsinto viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,clectroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposomemediated transfection(Dzau et al., Trends in Biotechnology 11, 205-210 [1993]). In somesituations it is desirable to provide the nucleic acid source with anagent that targets the target cells, such as an antibody specific for acell surface membrane protein or the target cell, a ligand for areceptor on the target cell, etc. Where liposomes are employed, proteinswhich bind to a cell surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake, e.g.capsid proteins or fragments thereof tropic for a particular cell type,antibodies for proteins which undergo internalization in cycling,proteins that target intracellular localization and enhanceintracellularhalf-life. The technique of receptor-mediatedendocytosis isdescribed, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414(1990). For review of the currently known gene marking and gene therapyprotocols see Anderson et al., Science 256, 808-813 (1992).

The I-TRAF polypeptides are useful in assays for identifying leadcompounds for therapeuticallyactive agents that modulate I-TRAF/TRAFbinding. Specifically, lead compounds that either prevent the formationof I-TRAF/TRAF complexes or prevent or inhibit dissociation of theI-TRAF/TRAF complexes already formed can be conveniently identified.Molecules preventing the interaction of I-TRAF and TRAF may find utilityunder circumstances when boosting of the immune system is desirable.Inhibitors of the dissociation of I-TRAF/TRAF complexes may be useful asimmunosuppressants or antiinflammatory agents. Screening assays can alsobe designed to find lead compounds that mimic the biological activity ofa native ligand of a TNF receptor superfamily member with which a TRAFprotein is associated, e.g. TNF, CD40 ligand, CD30 ligand, etc. Thesescreening methods involve assaying the candidate agents for theirability to release TRAFs from I-TRAF inhibition.

The screening assays of the present invention are amenable tohigh-throughput screening of chemical libraries, and are particularlysuitable for identifying small molecule drug candidates. Smallmolecules, which are usually less than 10 K molecular weight, aredesirable as therapeutics since they are more likely to be permeable tocells, are less susceptible to degradation by various cellularmechanisms, and are not as apt to elicit an immune response as proteins.Small molecules include but are not limited to synthetic organic orinorganic compounds. Many pharmaceutical companies have extensivelibraries of such molecules, which can be conveniently screened by usingthe assays of the present invention.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, cell based assays, etc. Such assay formats are well knownin the art.

The assay mixture typically contains an I-TRAF and a TRAF protein withwhich I-TRAF is normally associated (e.g. TRAF2 or TRAF3), usually in anisolated, partially pure or pure form. One or both of these componentsmay be fused to another peptide or polypeptide, which may, for example,provide or enhance protein-proteinbinding, improve stability under assayconditions, etc. In addition, one of the components usually comprises oris coupled to a detectable label. The label may provide for directdetection by measuring radioactivity, luminescence, optical or electrondensity, etc., or indirect detection such as an epitope tag, an enzyme,etc. The assay mixture additionally comprises a candidatepharmacological agent, and optionally a variety of other components,such as salts, buffers, carrier proteins, e.g. albumin, detergents,protease inhibitors, nuclease inhibitors, antimicrobial agents, etc.,which facilitate binding, increase stability, reduce non-specific orbackground interactions, or otherwise improve the efficiency orsensitivity of the assay.

To screen for inhibitors of I-TRAF/TRAF binding, the assay mixture isincubated under conditions whereby, but for the presence of thecandidate pharmacological agent, the I-TRAF specifically binds the TRAFprotein with a reference binding affinity. The mixture components can beadded in any order that provides for the requisite binding. Incubationmay be performed at any temperature which facilitates optimal binding,typically between about 4° and 40° C., more commonly between about 15°and 40° C. Incubation periods are likewise selected for optimal bindingbut also minimized to facilitate rapid, high-throughput screening, andare typically between about 0.1 and 10 hours, preferably less than 5hours, more preferably less than 2 hours. After incubation, the effectof the candidate pharmacological agent on the I-TRAF/TRAF binding isdetermined in any convenient way. For cell-free binding-type assays, aseparation step is often used to separate bound and unbound components.Separation may, for example, be effected by precipitation (e.g. TCAprecipitation, immunoprecipitation, etc.), immobilization (e.g. on asolid substrate), followed by washing. The bound protein is convenientlydetected by taking advantage of a detectable label attached to it, e.g.by measuring radioactive emission, optical or electron density, or byindirect detection using, e.g. antibody conjugates.

Compounds which inhibit or prevent the dissociation of the I-TRAF/TRAFcomplexes can be conveniently identified by forming the I-TRAF/TRAFcomplex in the absence of the candidate pharmacological agent, addingthe agent to the mixture, and changing the conditions such that, but forthe presence of the candidate agent, TRAF would be released from thecomplex. This can, for example, be achieved by changing the incubationtemperature or by adding to the mixture a compound which, in the absenceof the candidate pharmacological agent, would release TRAF from itscomplexed form. As an example, this compound can be a native ligand thesignal transduction of which is mediated by TRAF, e.g. TNF, a CD40ligand, a CD30 ligand, etc. The concentration of the free or bound TRAFcan then be detected and/or the dissociation constant of the I-TRAF/TRAFcomplex can be determined and compared with that of the control.

In order to identify lead compounds for therapeutically active agentsthat mimic the biological activity of a native ligand of a TNF receptorsuperfamily member with which a TRAF protein is associated (e.g. TNF,CD40 ligand, CD30 ligand, etc.), the candidate agent is added to amixture of I-TRAF and TRAF (e.g. TRAF2 or TRAF3). The mutual ratio ofthe TRAF protein and the cellular protein and the incubation conditionsare selected such that I-TRAF/fRAF complexes are formed prior to theaddition of the candidate agent. Upon addition of a candidate agent, itsability to release TRAF from the I-TRAF/TRAF complex is tested. Thetypical assay conditions, e.g. incubation temperature, time, separationand detection of bound and unbound material, etc. are as hereinabovedescribed. In a particular version of this assay, the assay mixtureadditionally contains a cellular protein with which the TRAF is normallyassociated (e.g. TNF-R2 or CD40), and the mutual ratio of the TRAFprotein and the cellular protein and the incubation conditions areselected such that TRAF signaling does not occur prior to the additionof the candidate ligand analog. Upon addition of the candidate molecule,its ability to initiate a TRAF-mediated signaling event is detected. Asan end point, it is possible to measure the ability of a candidate agentto induce TRAF-mediated NF-κB activation in a conventional manner.According to a preferred method, an NF-κB-dependent reporter gene, suchas an E-selectin-luciferase reporter construct (Schindler and Baichwal,Mol. Cell. Biol. 14, 5820 [1994]), is used in a cell type assay.Luciferase activities are determined and normalized based onβ-galactosidase expression. Alternatively, NF-κB activation can beanalyzed by electrophoretic mobility shift assay (Schutze et al., Cell71, 765-776 [1992]). However, other conventional biochemical assays areequally be suitable for detecting the release of TRAF from its complexed(and inhibited) form.

Based upon their ability to bind TRAFs, the I-TRAF polypeptides of thepresent invention can be used to purify native and variant TRAFs andtheir functional derivatives, which, in turn, are useful for thepurification of TNF-R2, CD40 and other members of the TNF receptorsuperfamily to which they specifically bind. Members of the TNF receptorsuperfamily are promising candidates for the treatment of a variety ofpathological conditions, for example, TNF-R2 (either as a solubleprotein or as a TNF-R2-Ig immunoadhesin) is in clinical trials for thetreatment of endotoxic (septic shock) and rheumatoid arthritis (RA).

The I-TRAF molecules of the present invention may additionally be usedto generate blocking (antagonist) or agonist anti-I-TRAF antibodies,which block or mimic the ability of I-TRAF to inhibit TRAF-mediatedsignal transduction. Generic methods for generating antibodies are wellknown in the art and are, for example, described in the textbooks andother literature referenced hereinbefore in connection with thedefinition of antibodies and concerning general techniques ofrecombinant DNA technology.

The I-TRAF molecules (including functional derivatives of nativeI-TRAFs) are also useful as therapeutics for the treatment ofpathological conditions associated with downstream effects of theoncogene LMP1 on cell growth and gene expression. LMP1 is anEpstein-Barr virus transforming protein which is believed to play animportant role in the development of tumors associated with EB virus,such as Burkitt's lymphoma (cancer of B lymphocytes) which has highincidence in West Africa and Papua New Guinea, and nasopharyngealcarcinoma, which is most common in Southern China and Greenland. Basedupon their ability to block TRAF-mediated signaling through LMP1, theI-TRAFs of the present invention may inhibit the downstream effects ofLMP1, including LMP1-mediated NF-κB activation, and are, optionally incombination with other cancer therapies, promising agents for thetreatment of these types of cancers.

The in vivo efficacy of the treatment of the present invention can bestudied against chemically induced tumors in various rodent models.Tumor cell lines propagated in in vitro cell cultures can be introducedin experimental rodents, e.g. mice by injection, for example by thesubcutaneous route. Techniques for chemical inducement of tumors inexperimental animals are well known in the art.

The treatment of the present invention may be combined with known tumortherapies, such as radiation therapy, chemotherapy, and immunotoxintherapy.

Therapeutic compositions comprising the I-TRAF polypeptides of thepresent invention, including functional derivatives, are prepared forstorage by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th Edition, Osol, A.Ed. 1980) in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients or stabilizers are nontoxic torecipients at the dosages and concentrationsemployed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid; low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,aminoacids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides,disaccharides and other carbohydrates including glucose,mannose, or dextrins; chelating agents such as EDTA; sugar alcohols suchas mannitol or sorbitol; salt-forming counterions such as sodium; and/ornonionic surfactants such as Tween, Pluronics or PEG.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsulesand poly-(methylmethacylate)microcapsulcs,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Suitable examples of sustained release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (U. Sidman et al.,Biopolymers 22 (1): 547-556 [1983]), poly (2-hydroxyethyl-methacrylate)(R. Langer, et al., J. Biomed. Mater. Res. 15: 167-277 [1981] and R.Langer, Chem. Tech. 12: 98-105 [1982]), ethylene vinyl acetate (R.Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).Sustained release compositions also include liposomes. Liposomescontaining a molecule within the scope of the present invention areprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Natl.Acad. Sci. USA 77: 4030-4034 (1980); EP 52322; EP 36676A; EP 88046; EP143949; EP 142641; Japanese patent application 83-118008; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are ofthe small (about 200-800 Angstroms) unilamelar type in which the lipidcontent is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal NT-4 therapy.

An effective amount of the active ingredient will depend, for example,upon the therapeutic objectives, the route of administration, and thecondition of the patient. Accordingly, it will be necessary for thetherapist to titer the dosage and modify the route of administration asrequired to obtain the optimal therapeutic effect. A typical dailydosage might range from about 1 μg/kg to up to 100 mg/kg or more,depending on the factors mentioned above. Typically, the clinician willadminister a molecule of the present invention until a dosage is reachedthat provides the required biological effect. The progress of thistherapy is easily monitored by conventional assays.

Further details of the invention will be apparent from the followingnon-limiting examples.

EXAMPLE 1

Cloning of Murine and Human I-TRAFs

I-TRAF was identified in a yeast two-hybrid screening system (Fields andSong, Nature 340, 245-246 [1989]) as described (Rothe et al., Cell 78681-692 [1994]), using full length TRAF2 in the GAL4 DNA-binding domainvector pPC97 (Chevray and Nathans, Proc. Natl. Acad. Sci. USA 89,5789-5793 [1992]) as bait. A plasmid cDNA library in theGAL4transcriptional activation domain vector pPC86 (Chevray and Nathans,supra) was constructed from SalI-NotI-adapted, double-stranded fetalliver stromal cell line 7-4 cDNA (FL, Rothe et al., supra) and murineperipheral lymph nodes (PLN; provided by P. Young, D. Dowbenko, and L.Lasky; U.S. Pat. No. 5,304,640 issued Apr. 19, 1994). Transformationefficiencies for the two libraries were 10 and 1 million, respectively.Restriction mapping of 24 positive clones indicated that most werederived from the same gene. Four FL and two PLN cDNA clones weresequenced on a 373A automated DNA sequencer (Applied Biosystems). The 5'ends of six additional murine I-TRAF DNA clones isolated by screening aCT6 cDNA library prepared in λgt22a were also sequenced (Rothe et al.,supra). The isolated cDNAs corresponded to several distinct transcriptsof the murine I-TRAF gene. Due to alternative splicing, thesetranscripts have the potential to utilize two different translationinitiation codons. The two major forms of murine I-TRAF mRNA arepredicted to encode proteins of 413 and 447 amino acids that we havetermed I-TRAFα and I-TRAFβ, respectively (FIG. 1a; SEQ. ID. NOs: 1 and2).

Five human I-TRAF cDNA clones, obtained by screening a λgt11 HUVEC cDNAlibrary (Hsu et al., Cell 81, 495-504 [1995]) with a murine I-TRAFprobe, were sequenced. All isolated human I-TRAF cDNAs encode a fulllength 425 amino acid protein (M_(r) 48 K) that shows a 82% amino acididentity to murine I-TRAFα (FIG. 1a). Database searches failed to revealany proteins having significant sequence similarity to I-TRAF. Northernblot analysis using mRNA from mouse tissues indicated that the ˜2.4 kbI-TRAF mRNA is ubiquitously expressed (FIG. 1b). The multiple tissueblot (Clontech) was hybridized with a murine I-TRAF cDNA probe accordingto the Clontech protocol. I-TRAF mRNA is the band at ˜2.4 kb.

Murine and human I-TRAF cDNA sequences have been deposited in GenBankand in the American Type Culture Collection (ATCC), 12301 ParklawnDrive, Rockville, Md., USA, under accession numbers and, respectively.

EXAMPLE 2

Characterization of I-TRAF

(1) Two-hybrid binding assay

Inspection of the various I-TRAF cDNAs obtained by two-hybrid screeningindicated that the N-terminal portion of murine I-TRAF (amino acids35-236) is sufficient for interaction with TRAF2. Further two-hybridexperiments were performed to determine which portion of TRAF2 isrequired for interaction with I-TRAF. Yeast HF7c cells werecotransformed with an expression vector encoding a Gal 14 activationdomain-I-TRAF fusion protein and the indicated Gal4 DNA-binding domainexpression vectors (Rothe et al., supra). Each transformation mixturewas plated on a synthetic dextrose plate lacking leucine and tryptophan.Filter assays for β-galactosidase activity were performed to detectinteraction between fusion proteins (Fields and Song, supra). The dataare presented in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        DNA-binding    Activation                                                                                domain hybrid domain hybrid Interaction*           ______________________________________                                        --             I-TRAF     -                                                     TRAF1 I-TRAF ++                                                               TRAF2 I-TRAF ++                                                               TRAF2 (87-501) I-TRAF ++                                                      TRAF3 I-TRAF +                                                                TNF-R2 I-TRAF -                                                             ______________________________________                                    

The results show that only the conserved TRAF domain (amino acids264-501) of TRAF2 is required for interaction with I-TRAF. Furthermore,both TRAF1 and TRAF3 also associate with I-TRAF.

(2) In vitro binding assay

The interactions of ³⁵ S-labeled TRAF1 (Rothe et al., supra), TRAF2(Rothe et al., supra), TRAF3 (Hu et al., supra), TNF-R2 (Smith et al.,Science 248, 1019-1023 [1990]), and TRADD (Hsu et al., Cell 81, 495-504[1995]) with I-TRAFα expressed as a glutathione-S-transferase(GST)fusion protein (GST-I-TRAF)and control GST protein were examined.GST-I-TRAF was expressed using the pGEX3X vector (Pharmacia) andpurified as described (Smith and Johnson, Gene 67, 31-40 [1988]). ³⁵S-labeled proteins were generated using TNT coupled reticulocyte lysatesystem (Promega) and the various cDNAs cloned in pBluescript KS(Stratagene) or pRK5 (Schall et al., Cell 61, 361-370 [1990]). For eachbinding assay, 0.5 μg GST-I-TRAF or 0.5 μg GST bound to glutathioneSepharose beads was incubated with equivalent cpm of the individual ³⁵S-labeled proteins in 1 ml of E1A buffer (50 mM HEPES [pH 7.6], 250 mMNaCl, 0.1% NP-40, 5 mM EDTA) at 4° C. for 1 hour. Beads were washed sixtimes with E1A buffer and precipitates were fractionated by 10%SDS-PAGE. The gel was dried and exposed to Kodak X-ray film. By thisassay, I-TRAF was found to specifically interact with TRAF1, TRAF2, andTRAF3.

Results from both two-hybrid and in vitro binding experiments show thatI-TRAF interacts more strongly with TRAF1 and TRAF2 than with TRAF3.

(3) Three-hybrid interaction test

I-TRAF does not directly interact with TNF-R2 (Table 1). However, asTRAF1 and TRAF2 form a complex with TNF-R2, it was important to ask ifI-TRAF can indirectly associate with TNF-R2 via TRAFs. A three-hybridinteraction test (Rothe et al., supra) was performed to address thisquestion. Whereas TRAF2 can bind simultaneously to TRAF1 and TNF-R2(Rothe et al., supra), it was not able to mediate I-TRAF interactionwith TNF-R2. In fact, I-TRAF expression in yeast was found to inhibitthe association of TRAF2 with TNF-R2 (data not shown). This result isconsistent with I-TRAF and TNF-R2 both binding to the same C-terminalTRAF domain of TRAF2.

(4) Inhibition of TRAF2:TNF-R2 interaction in mammalian cells

The observed inhibitory effect of I-TRAF on the TRAF2:TNF-R2 interactionwas further investigated in mammalian cells. TRAF2 and I-TRAF wereexpressed as full length proteins containing N-terminal FLAG epitopetags (Kodak) using the cytomegalovirus-based expression vector pRK5(Schall et al., supra). Human embryonic kidney 293 cells (2×10⁶) wereseeded on 150 mm dishes and transfected (Ausubel et al., Curr. Prot.Mol. Biol. 1, 9.1.1-9.1.3 [1994]) the following day with pRK-TRAF2 (0 or1 μg) expression vector (Rothe et al., Science in press; copendingapplication Ser. No. 08/446,915 filed May 22, 1995) and increasingamounts of pRK-FLAG-I-TRAF (0, 1, 3, or 9 μg) expression vector. Cellswere incubated at 37° C. for 24 hours, and lysed in 0.5 ml E1A buffer.450 μl aliquots of the lysates were incubated with 10 μl GST-TNF-R2fusion protein (Rothe et al., Cell supra; copending application Ser.No.08/446,915) bound to glutathione Sepharose beads for 15 minutes at 4°C. The beads were washed three times with 0.6 ml E1A buffer and boundproteins fractionated by 8% SDS-PAGE and transferred to a nitrocellulosemembrane. Western blot analysis was performed using an anti-FLAGmonoclonal antibody M2 (Kodak) and horseradish peroxidase-coupledrabbitanti-mouse immunoglobulin(Amersham). Detection was by enhancedchemiluminescenceaccording to the Amersham protocol. Western blotanalysis of 50 μl aliquots of the lysates indicated that TRAF2expression levels were similar in all samples transfected with pRK-TRAF2(data not shown). Coexpression of TRAF2 with increasing amounts ofI-TRAF effectively blocked the TRAF2:TNF-R2 interaction (FIG. 3).Furthermore, no I-TRAF was precipitated by the TRAF2:TNF-R2 complex.These results show that TRAF2 us not able to bind TNF-R2 and I-TRAFsimultaneously, possibly due to binding sites that are overlapping.

The ability of I-TRAF to bind the three known TRAF family members raisedthe intriguing possibility that I-TRAF functions as a general regulatorof TRAF-mediated signaling events. Furthermore, the observed inhibitoreffect of I-TRAF on TRAF2:TNF-R2 interaction suggested that I-TRAFexpression might negatively influence TRAF2 signal transduction.Consequently, we measured the effect of I-TRAF expression onTRAF2-mediated NF-κB activation.

(5) The inhibitory effect of I-TRAF on activation of NF-κB

Human embryonic kidney 293 cells were seeded at 2×10⁵ cells/well on6-well (35 mm) dishes and transfected (Ausubel el al., Curr. Prot. Mol.Biol. 1, 9.1.1-0.1.3 [1994]) the following day with (a) 0.5 μg pRK-TRAF2(Rothe et al., Science in press; copending application Ser. No.08/446,915) (b) 1 μg pCDM8-CD40 (Rothe et al., Science supra; copendingapplication Ser. No. 08/446,915), or (c) 0.1 μg pRK5mTNF-R2 (Lewis etal., Proc. Natl. Acad. Sci. USA 88, 2830-2834 [1991]), and increasingamounts of pRK-I-TRAF (0, 0.1, 0.32, 1.0 and 3.0 μg). Each transfectionalso contained 0.25 μg of pELAM-luc reported plasmid and 1 μg pRSV-βgal(Hsu et al., supra). For each of (a), (b) and (c), a controltransfection containing only the pELAM-luc and pRSV-βgal was performed.After 24 hours, luciferase activities and normalizations toβ-galactosidase levels were performed as described (Hsu et al., supra).Values relative to control are shown in FIG. 4 as mean±SEM forexperiments in which each transfection was performed in triplicate.

Transient expressionof TRAF2 in 293 cells potently activates theNF-κB-dependentreported gene. (Rothe et al., Science supra). Thisactivation was dramatically inhibited by I-TRAF coexpression in adose-dependent manner (FIG. 4a). We next examined the effect of I-TRAFoverexpressionon NF-κB activation triggered by the TRAF2-interactingreceptors CD40 and TNF-R2. Transient expression of thesereceptors has been shown to induce ligand-independent receptoraggregation which activated NF-κB in a TRAF2-dependent process. (Rotheet al., Science supra). As observed above for TRAF2, NF-κB activationthrough both CD40 (FIG. 4b) and TNF-R2 (FIG. 4c) was effectively blockedby increased expression of I-TRAF. NF-κB activation mediated byinterleukin 1 (IL-1) was only slightly depressed by I-TRAFoverexpression (data not shown). Thus, I-TRAF is a specific inhibitor ofTRAF2-dependent NF-κB activation signaled by CD40 and TNF-R2.

(6) Conclusions

Conceptually, the I-TRAF/TRAF system bears remarkable similarity to theextensively studied IκB/NF-κB regulatory system (Liou and Baltimore,Curr. Op. Cell. Bio. 5, 477-487 [1993]; Thanos & Maniatis, Cell 80,529-532 [1995]). Both systems are involved in regulating the activity ofthe transcription factor NF-κB through the utilization of inhibitoryproteins. IκB directly regulates NF-κB function by keeping itsequestered in the cytoplasm. I-TRAF acts at an earlier step by bindingto TRAFs and preventing their interaction with cell surface receptors(CD40, TNF-R2) that trigger TRAF2-mediated NF-κB activation cascades.The function of I-TRAF may be to prevent continuous TRAF signaling inthe absence of ligand (CD40L or TNF). This function is consistent withthe finding that TRAF2 overexpressionalone, in the absence of receptoraggregation, can trigger NF-κB activation. Ligand-induced receptoraggregation might be expected to release TRAFs from I-TRAF inhibition byproviding a new, higher affinity TRAF binding site.

All documents cited throughout the specification and all referencescited therein are hereby expressly incorporated by reference. Althoughthe invention has been described with reference to certain embodiments,it will be understood that certain variations and modification arepossible and will be readily available for those of ordinary skill inthe art. All such changes and modification and with the scope of thepresent invention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 8                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 413 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Met Asp Lys Asn Ile Gly Glu Gln Leu Asn Ar - #g Ala Tyr Glu Ala           1               5 - #                 10 - #                 15              - - Phe Arg Gln Ala Cys Met Asp Arg Asp Ser Al - #a Val Arg Glu Leu                           20 - #                 25 - #                 30              - - Gln Gln Lys Thr Glu Asn Tyr Glu Gln Arg Il - #e Arg Glu Gln Gln                           35 - #                 40 - #                 45              - - Glu Gln Leu Ser Phe Gln Gln Asn Leu Ile As - #p Arg Leu Lys Ser                           50 - #                 55 - #                 60              - - Gln Leu Leu Leu Val Asp Ser Ser Arg Asp As - #n Ser Tyr Gly Tyr                           65 - #                 70 - #                 75              - - Val Pro Leu Leu Glu Asp Ser Asp Arg Arg Ly - #s Asn Asn Leu Thr                           80 - #                 85 - #                 90              - - Leu Asp Glu Pro His Asp Lys Val Lys Leu Gl - #y Thr Leu Arg Asp                           95 - #                100 - #                105              - - Lys Gln Ser Lys Val Arg Arg Gln Glu Val Se - #r Ser Gly Lys Glu                          110  - #               115  - #               120              - - Ser Ala Lys Gly Leu Asn Ile Pro Leu His Hi - #s Glu Arg Asp Asn                          125  - #               130  - #               135              - - Ile Glu Lys Thr Phe Trp Asp Leu Lys Glu Gl - #u Phe His Arg Ile                          140  - #               145  - #               150              - - Cys Leu Leu Ala Lys Ala Gln Lys Asp His Le - #u Ser Lys Leu Asn                          155  - #               160  - #               165              - - Ile Pro Asp Ile Ala Thr Asp Thr Gln Cys Se - #r Val Pro Ile Gln                          170  - #               175  - #               180              - - Cys Thr Asp Lys Thr Glu Lys Gln Glu Ala Le - #u Phe Lys Pro Gln                          185  - #               190  - #               195              - - Ala Lys Asp Asp Ile Asn Arg Gly Met Ser Cy - #s Val Thr Ala Val                          200  - #               205  - #               210              - - Thr Pro Arg Gly Leu Gly Arg Asp Glu Glu As - #p Thr Ser Phe Glu                          215  - #               220  - #               225              - - Ser Leu Ser Lys Phe Asn Val Lys Phe Pro Pr - #o Met Asp Asn Asp                          230  - #               235  - #               240              - - Ser Ile Phe Leu His Ser Thr Pro Glu Ala Pr - #o Ser Ile Leu Ala                          245  - #               250  - #               255              - - Pro Ala Thr Pro Glu Thr Val Cys Gln Asp Ar - #g Phe Asn Met Glu                          260  - #               265  - #               270              - - Val Arg Asp Asn Pro Gly Asn Phe Val Lys Th - #r Glu Glu Thr Leu                          275  - #               280  - #               285              - - Phe Glu Ile Gln Gly Ile Asp Pro Ile Thr Se - #r Ala Ile Gln Asn                          290  - #               295  - #               300              - - Leu Lys Thr Thr Asp Lys Thr Asn Pro Ser As - #n Leu Arg Ala Thr                          305  - #               310  - #               315              - - Cys Leu Pro Ala Gly Asp His Asn Val Phe Ty - #r Val Asn Thr Phe                          320  - #               325  - #               330              - - Pro Leu Gln Asp Pro Pro Asp Ala Pro Phe Pr - #o Ser Leu Asp Ser                          335  - #               340  - #               345              - - Pro Gly Lys Ala Val Arg Gly Pro Gln Gln Pr - #o Phe Trp Lys Pro                          350  - #               355  - #               360              - - Phe Leu Asn Gln Asp Thr Asp Leu Val Val Pr - #o Ser Asp Ser Asp                          365  - #               370  - #               375              - - Ser Glu Leu Leu Lys Pro Leu Val Cys Glu Ph - #e Cys Gln Glu Leu                          380  - #               385  - #               390              - - Phe Pro Pro Ser Ile Thr Ser Arg Gly Asp Ph - #e Leu Arg His Leu                          395  - #               400  - #               405              - - Asn Thr His Phe Asn Gly Glu Thr                                                          410  - #       413                                             - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1955 base - #pairs                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - CTGGAACGGA AAGCTACTTC CGGTTGCAGT CATTCTGCCG GGCACCGGCG  - #                  50                                                                         - - ACCTGTGGCG TGAGCGAGCA CAGCCGGAAC CCTCCACTAG CTGGCATTCC  - #                 100                                                                         - - TACCATCCTT TATAGTGATG CTACAGGACA AAGAGGAATG GATAAAAACA  - #                 150                                                                         - - TTGGTGAGCA ACTCAATAGA GCATATGAAG CCTTCCGACA GGCATGCATG  - #                 200                                                                         - - GATAGAGATT CAGCAGTAAG AGAGCTACAG CAAAAGCAGA CTGAGAACTA  - #                 250                                                                         - - TGAACAAAGA ATACGCGAGC AACAGGAACA GCTGTCATTT CAACAAAACC  - #                 300                                                                         - - TAATTGACAG GCTGAAATCA CAGCTACTTC TCGTGGATTC TAGTCGAGAT  - #                 350                                                                         - - AACAGTTATG GCTATGTACC TTTGCTTGAA GACAGTGACA GAAGGAAGAA  - #                 400                                                                         - - TAATTTGACC CTTGATGAAC CACATGATAA AGTGAAACTA GGAACACTGA  - #                 450                                                                         - - GAGATAAGCA ATCAAAGGTG AGACGACAAG AAGTTTCTTC TGGAAAAGAA  - #                 500                                                                         - - TCCGCCAAGG GTCTCAACAT CCCTCTGCAT CACGAAAGGG ATAATATAGA  - #                 550                                                                         - - GAAGACTTTC TGGGACCTTA AAGAAGAATT TCATAGGATT TGCTTGCTAG  - #                 600                                                                         - - CAAAAGCACA GAAAGATCAC TTAAGCAAAC TTAATATACC AGATATTGCA  - #                 650                                                                         - - ACTGACACAC AGTGTTCTGT GCCTATACAG TGTACTGATA AAACAGAGAA  - #                 700                                                                         - - ACAAGAAGCG CTGTTTAAGC CCCAGGCTAA AGATGATATA AATAGAGGTA  - #                 750                                                                         - - TGTCGTGCGT CACAGCTGTC ACACCAAGAG GACTGGGCCG GGATGAGGAA  - #                 800                                                                         - - GATACCTCTT TTGAATCACT TTCTAAATTC AATGTCAAGT TTCCGCCTAT  - #                 850                                                                         - - GGACAATGAC TCTATTTTTC TACATAGCAC TCCAGAGGCC CCGAGCATCC  - #                 900                                                                         - - TTGCTCCTGC CACACCTGAG ACAGTGTGCC AGGACCGATT TAATATGGAA  - #                 950                                                                         - - GTCAGAGACA ACCCAGGAAA CTTTGTTAAA ACAGAAGAAA CTTTATTTGA  - #                1000                                                                         - - AATTCAGGGA ATTGACCCCA TAACTTCAGC TATACAAAAC CTTAAAACAA  - #                1050                                                                         - - CTGACAAAAC AAACCCCTCA AATCTTAGAG CGACGTGTTT GCCAGCTGGA  - #                1100                                                                         - - GACCACAATG TGTTCTATGT AAATACGTTC CCACTTCAAG ACCCGCCTGA  - #                1150                                                                         - - CGCACCTTTT CCCTCACTGG ATTCCCCAGG AAAGGCTGTC CGAGGACCAC  - #                1200                                                                         - - AGCAGCCCTT TTGGAAGCCT TTTCTTAACC AAGACACTGA CTTAGTGGTA  - #                1250                                                                         - - CCAAGTGATT CAGACTCAGA GCTCCTTAAA CCTCTAGTGT GTGAATTCTG  - #                1300                                                                         - - TCAAGAGCTT TTCCCACCAT CCATTACATC CAGAGGGGAT TTCCTCCGGC  - #                1350                                                                         - - ATCTTAATAC ACACTTTAAT GGGGAGACTT AAATCACGTT TGAAAACAGA  - #                1400                                                                         - - CATATCATGT TCTCTGTGGT GGTTTTGGAT TTGTAACGCT AGAGAACGCT  - #                1450                                                                         - - TTCTCGTGAG CCAAATGTAA GATTGATTAT AAAGTTGTTA CTTTATCTTT  - #                1500                                                                         - - TAAGAGATCA TTTTGTATAG AACTATAACT CATTATATTA TTCATGTTTA  - #                1550                                                                         - - TACCTATAAT TTCTACATTT CAAAATTACA CATGTGACTT ACAGAGTTAT  - #                1600                                                                         - - TCAGTCATAA TTTATGTTTC AAATAGCTAA GTTTATTGTT TGACTATTGT  - #                1650                                                                         - - GAGATCTATT AAATTTAGTA ATAGCAAATG TTTATAGGAT ATTCAAATTT  - #                1700                                                                         - - CATTTGAATT TTTAATTATT TTTGCTACAG GTAATATTCC TTTAAAATAC  - #                1750                                                                         - - GTATATAACG TACAGAGAAT AACAGACAAT ATGATCTAAG TAAATGTCGA  - #                1800                                                                         - - ATCAATCATT AGTTGCCCAG GGAAATTTAA ACATTATAGA TCATTTTTAA  - #                1850                                                                         - - ATAATACACA TAGTTTTAAT TTTTACTGTG TGTATAGATG CATGATTAAA  - #                1900                                                                         - - TGACTTAAAT ATTAAAAGTG ACTTACGTCG TGCTTATTAA AAAAAAAAAA  - #                1950                                                                         - - AAAAA                 - #                  - #                  - #              1955                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Met Ser Leu Lys Arg His Ser Leu Arg Arg As - #n Ala Cys His Leu            1               5 - #                 10 - #                 15              - - Glu Thr Arg Ala Gly Ile Pro Thr Ile Leu Ty - #r Ser Asp Ala Thr                           20 - #                 25 - #                 30              - - Gly Gln Arg Gly                                                                       34                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 170 base - #pairs                                                 (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CCCACGCGTC CGGTTTGGGC AGCATCTGTA GAGCCTGTGC AAACGGCTTC  - #                  50                                                                         - - CAGAATGGGT ACGTGCCTAT GTCTTTAAAG AGACATAGTC TGCGAAGGAA  - #                 100                                                                         - - CGCCTGTCAC CTGGAGACGA GAGCTGGCAT TCCTACCATC CTTTATAGTG  - #                 150                                                                         - - ATGCTACAGG ACAAAGAGGA            - #                  - #                      - #170                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 425 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Met Asp Lys Asn Ile Gly Glu Gln Leu Asn Ly - #s Ala Tyr Glu Ala            1               5 - #                 10 - #                 15              - - Phe Arg Gln Ala Cys Met Asp Arg Asp Ser Al - #a Val Lys Glu Leu                           20 - #                 25 - #                 30              - - Gln Gln Lys Thr Glu Asn Tyr Glu Gln Arg Il - #e Arg Glu Gln Gln                           35 - #                 40 - #                 45              - - Glu Gln Leu Ser Leu Gln Gln Thr Ile Ile As - #p Lys Leu Lys Ser                           50 - #                 55 - #                 60              - - Gln Leu Leu Leu Val Asn Ser Thr Gln Asp As - #n Asn Tyr Gly Cys                           65 - #                 70 - #                 75              - - Val Pro Leu Leu Glu Asp Ser Asp Thr Arg Ly - #s Asn Thr Leu Thr                           80 - #                 85 - #                 90              - - Leu Ala Gln Pro Gln Asp Lys Val Ile Ser Gl - #y Ile Ala Arg Glu                           95 - #                100 - #                105              - - Lys Leu Pro Lys Val Arg Arg Gln Glu Val Se - #r Ser Pro Arg Lys                          110  - #               115  - #               120              - - Glu Thr Ser Ala Arg Ser Leu Gly Ser Pro Le - #u Leu His Glu Arg                          125  - #               130  - #               135              - - Gly Asn Ile Glu Lys Thr Ser Trp Asp Leu Ly - #s Glu Glu Phe His                          140  - #               145  - #               150              - - Lys Ile Cys Met Leu Ala Lys Ala Gln Lys As - #p His Leu Ser Lys                          155  - #               160  - #               165              - - Leu Asn Ile Pro Asp Thr Ala Thr Glu Thr Gl - #n Cys Ser Val Pro                          170  - #               175  - #               180              - - Ile Gln Cys Thr Asp Lys Thr Asp Lys Gln Gl - #u Ala Leu Phe Thr                          185  - #               190  - #               195              - - Pro Gln Ala Lys Asp Asp Ile Asn Arg Gly Al - #a Pro Ser Ile Thr                          200  - #               205  - #               210              - - Ser Val Thr Pro Arg Gly Leu Cys Arg Asp Gl - #u Glu Asp Thr Ser                          215  - #               220  - #               225              - - Leu Glu Ser Leu Ser Lys Phe Asn Val Lys Ph - #e Pro Pro Met Asp                          230  - #               235  - #               240              - - Asn Asp Ser Thr Phe Leu His Ser Thr Pro Gl - #u Arg Pro Gly Ile                          245  - #               250  - #               255              - - Leu Ser Pro Ala Thr Ser Glu Ala Val Cys Gl - #n Glu Lys Phe Asn                          260  - #               265  - #               270              - - Met Glu Phe Arg Asp Asn Pro Gly Asn Phe Va - #l Lys Thr Glu Glu                          275  - #               280  - #               285              - - Thr Leu Phe Glu Ile Gln Gly Ile Asp Pro Il - #e Ala Ser Ala Ile                          290  - #               295  - #               300              - - Gln Asn Leu Lys Thr Thr Asp Lys Thr Lys Pr - #o Ser Asn Leu Val                          305  - #               310  - #               315              - - Asn Thr Cys Ile Arg Thr Thr Leu Asp Arg Al - #a Ala Cys Leu Pro                          320  - #               325  - #               330              - - Pro Gly Asp His Asn Ala Leu Tyr Val Asn Se - #r Phe Pro Leu Leu                          335  - #               340  - #               345              - - Asp Pro Ser Asp Ala Pro Phe Pro Ser Leu As - #p Ser Pro Gly Lys                          350  - #               355  - #               360              - - Ala Ile Arg Gly Pro Gln Gln Pro Ile Trp Ly - #s Pro Phe Pro Asn                          365  - #               370  - #               375              - - Gln Asp Ser Asp Ser Val Val Leu Ser Gly Th - #r Asp Ser Glu Leu                          380  - #               385  - #               390              - - His Ile Pro Arg Val Cys Glu Phe Cys Gln Al - #a Val Phe Pro Pro                          395  - #               400  - #               405              - - Ser Ile Thr Ser Arg Gly Asp Phe Leu Arg Hi - #s Leu Asn Ser His                          410  - #               415  - #               420              - - Phe Asn Gly Glu Thr                                                                      425                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2108 base - #pairs                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GTTTGAGCAG CATTGTTAGA GCCTGTGGAA AACACTTTAC AACTGTGTAA  - #                  50                                                                         - - CTGTCTTCAT CTTTACAGAG GAATAGTCTA CAAAGGAAGA CTTGTAACCT  - #                 100                                                                         - - GGAGAAGAGA CCTGTCATTT ACTCCATCCT TTATAGTGAT GCTACAGGAC  - #                 150                                                                         - - GAAGAGGAAT GGATAAAAAC ATTGGCGAGC AACTCAATAA AGCGTATGAA  - #                 200                                                                         - - GCCTTCCGGC AGGCATGCAT GGATAGAGAT TCTGCAGTAA AAGAATTACA  - #                 250                                                                         - - GCAAAAGACT GAGAACTATG AGCAGAGAAT ACGTGAACAA CAGGAACAGC  - #                 300                                                                         - - TGTCACTCCA ACAGACTATT ATTGACAAGC TAAAATCTCA GTTACTTCTT  - #                 350                                                                         - - GTGAATTCCA CTCAAGATAA CAATTATGGC TGTGTCCCTC TGCTTGAAGA  - #                 400                                                                         - - CAGTGACACA AGAAAGAATA CTTTGACTCT TGCTCAGCCA CAAGATAAAG  - #                 450                                                                         - - TGATTTCAGG AATAGCAAGA GAAAAACTAC CAAAGGTAAG AAGACAAGAG  - #                 500                                                                         - - GTTTCTTCTC CTAGAAAAGA AACTTCAGCA AGGAGTCTTG GCAGTCCTTT  - #                 550                                                                         - - GCTCCATGAA AGGGGTAATA TAGAGAAGAC TTCCTGGGAT CTGAAAGAAG  - #                 600                                                                         - - AATTTCATAA AATATGCATG CTAGCAAAAG CACAGAAAGA CCACTTAAGC  - #                 650                                                                         - - AAACTTAATA TACCAGACAC TGCAACTGAA ACACAGTGCT CTGTGCCTAT  - #                 700                                                                         - - ACAGTGTACG GATAAAACAG ATAAACAAGA AGCGCTGTTT ACGCCTCAGG  - #                 750                                                                         - - CTAAAGATGA TATAAATAGA GGTGCACCAT CCATCACATC TGTCACACCA  - #                 800                                                                         - - AGAGGACTGT GCAGAGATGA GGAAGACACC TCTTTGGAAT CACTTTCTAA  - #                 850                                                                         - - ATTCAATGTC AAGTTTCCAC CTATGGACAA TGACTCAACT TTCTTACATA  - #                 900                                                                         - - GCACTCCAGA GAGACCCGGC ATCCTTAGTC CTGCCACGTC TGAGGCAGTG  - #                 950                                                                         - - TGCCAAGAGA AATTTAATAT GGAGTTCAGA GACAACCCAG GGAACTTTGT  - #                1000                                                                         - - TAAAACAGAA GAAACTTTAT TTGAAATTCA GGGAATTGAC CCCATAGCTT  - #                1050                                                                         - - CAGCTATACA AAACCTTAAA ACAACTGACA AAACAAAGCC CTCAAATCTC  - #                1100                                                                         - - GTAAACACTT GTATCAGGAC AACTCTGGAT AGAGCTGCGT GTTTGCCACC  - #                1150                                                                         - - TGGAGACCAT AATGCATTAT ATGTAAATAG CTTCCCACTT CTGGACCCAT  - #                1200                                                                         - - CTGATGCACC TTTTCCCTCA CTCGATTCCC CGGGAAAAGC AATCCGAGGA  - #                1250                                                                         - - CCACAGCAGC CCATTTGGAA GCCCTTTCCT AATCAAGACA GTGACTCGGT  - #                1300                                                                         - - GGTACTAAGT GGCACAGACT CAGAACTGCA TATACCTCGA GTATGTGAAT  - #                1350                                                                         - - TCTGTCAAGC AGTTTTCCCA CCATCCATTA CATCCAGGGG GGATTTCCTT  - #                1400                                                                         - - CGGCATCTTA ATTCACACTT CAATGGAGAG ACTTAAGACA CATTTGAAAA  - #                1450                                                                         - - CAGACATATC AAGTTCTATG TGATGATTTT GGGTTTTTAA TACTATAAAT  - #                1500                                                                         - - ACTTGATTGT AAACTAAATT CAAGATCATT TATAGGAAAA TCTAGTTTCA  - #                1550                                                                         - - CAGCTATTTG AATTTTTTTC TGGATTTACT ATATAACTCT TATTTTTTAA  - #                1600                                                                         - - AAGATCATTC TGTTCTTTCA AGGAGAAATA AGCCTAAAAG AAGAAAAACA  - #                1650                                                                         - - AAAAAAATTC TGTATAAAAC TGTAATCCTT TGTATTCATG TTTACAGTGC  - #                1700                                                                         - - TATTACTATA ATTCAAAATT ATGTATGTGA CTTAGAGTTA TATAATCATA  - #                1750                                                                         - - ATTTATGTTT ATTTCAAATA TCTAAGTTTA TTGCTTGGAT TTCTAGTGAG  - #                1800                                                                         - - AGCTGTTGAA TTTGGTGATG TCAAATGTTT CTAGGGTTTT TTAGTTTGTT  - #                1850                                                                         - - TTTATTGAGA AAATTGATTA TTTATGCTAT AGGTGATATT CTCTTTGAAT  - #                1900                                                                         - - AAACCTATAA TAGGAAATAG CAGACCACAT AAACATCTTT GTAAATATCA  - #                1950                                                                         - - AACCTAATAC ATTTCTTGTC CAGTGATAAA ACAACTGGTA GAATTATTTA  - #                2000                                                                         - - AACACTTTAG ATTTTTAAAT AATAAACATG GCTTTAATTT TTACTGTGTG  - #                2050                                                                         - - TATAGCTACA TGATGAAATT AATTAAATAT TAAGAGGTAA AAAAAAAAAA  - #                2100                                                                         - - AAAAAAAA                - #                  - #                  -     #        2108                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1922 base - #pairs                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - CGGAAAGCTA CTTCCGGTTG CAGTCATTCT GCCGGGCACC GGCGACCTGT  - #                  50                                                                         - - GGCGTGAGCG AGCACAGCCG GAACCCTCCA CTAGCTGGCA TTCCTACCAT  - #                 100                                                                         - - CCTTTATAGT GATGCTACAG GACAAAGAGG AATGGATAAA AACATTGGTG  - #                 150                                                                         - - AGCAACTCAA TAGAGCATAT GAAGCCTTCC GACAGGCATG CATGGATAGA  - #                 200                                                                         - - GATTCAGCAG TAAGAGAGCT ACAGCAAAAG CAGACTGAGA ACTATGAACA  - #                 250                                                                         - - AAGAATACGC GAGCAACAGG AACAGCTGTC ATTTCAACAA AACCTAATTG  - #                 300                                                                         - - ACAGGCTGAA ATCACAGCTA CTTCTCGTGG ATTCTAGTCG AGATAACAGT  - #                 350                                                                         - - TATGGCTATG TACCTTTGCT TGAAGACAGT GACAGAAGGA AGAATAATTT  - #                 400                                                                         - - GACCCTTGAT GAACCACATG ATAAAGTGAA ACTAGGAACA CTGAGAGATA  - #                 450                                                                         - - AGCAATCAAA GGTGAGACGA CAAGAAGTTT CTTCTGGAAA AGAATCCGCC  - #                 500                                                                         - - AAGGGTCTCA ACATCCCTCT GCATCACGAA AGGGATAATA TAGAGAAGAC  - #                 550                                                                         - - TTTCTGGGAC CTTAAAGAAG AATTTCATAG GATTTGCTTG CTAGCAAAAG  - #                 600                                                                         - - CACAGAAAGA TCACTTAAGC AAACTTAATA TACCAGATAT TGCAACTGAC  - #                 650                                                                         - - ACACAGTGTT CTGTGCCTAT ACAGTGTACT GATAAAACAG AGAAACAAGA  - #                 700                                                                         - - AGCGCTGTTT AAGCCCCAGG CTAAAGATGA TATAAATAGA GGTATGTCGT  - #                 750                                                                         - - GCGTCACAGC TGTCACACCA AGAGGACTGG GCCGGGATGA GGAAGATACC  - #                 800                                                                         - - TCTTTTGAAT CACTTTCTAA ATTCAATGTC AAGTTTCCGC CTATGGACAA  - #                 850                                                                         - - TGACTCTATT TTTCTACATA GCACTCCAGA GGCCCCGAGC ATCCTTGCTC  - #                 900                                                                         - - CTGCCACACC TGAGACAGTG TGCCAGGACC GATTTAATAT GGAAGTCAGA  - #                 950                                                                         - - GACAACCCAG GAAACTTTGT TAAAACAGAA GAAACTTTAT TTGAAATTCA  - #                1000                                                                         - - GGGAATTGAC CCCATAACTT CAGCTATACA AAACCTTAAA ACAACTGACA  - #                1050                                                                         - - AAACAAACCC CTCAAATCTT AGAGCGACGT GTTTGCCAGC TGGAGACCAC  - #                1100                                                                         - - AATGTGTTCT ATGTAAATAC GTTCCCACTT CAAGACCCGC CTGACGCACC  - #                1150                                                                         - - TTTTCCCTCA CTGGATTCCC CAGGAAAGGC TGTCCGAGGA CCACAGCAGC  - #                1200                                                                         - - CCTTTTGGAA GCCTTTTCTT AACCAAGACA CTGACTTAGT GGTACCAAGT  - #                1250                                                                         - - GATTCAGACT CAGAGCTCCT TAAACCTCTA GTGTGTGAAT TCTGTCAAGA  - #                1300                                                                         - - GCTTTTCCCA CCATCCATTA CATCCAGAGG GGATTTCCTC CGGCATCTTA  - #                1350                                                                         - - ATACACACTT TAATGGGGAG ACTTAAATCA CGTTTGAAAA CAGACATATC  - #                1400                                                                         - - ATGTTCTCTG TGGTGGTTTT GGATTTGTAA CGCTAGAGAA CGCTTTCTCG  - #                1450                                                                         - - TGAGCCAAAT GTAAGATTGA TTATAAAGTT GTTACTTTAT CTTTTAAGAG  - #                1500                                                                         - - ATCATTTTGT ATAGAACTAT AACTCATTAT ATTATTCATG TTTATACCTA  - #                1550                                                                         - - TAATTTCTAC ATTTCAAAAT TACACATGTG ACTTACAGAG TTATTCAGTC  - #                1600                                                                         - - ATAATTTATG TTTCAAATAG CTAAGTTTAT TGTTTGACTA TTGTGAGATC  - #                1650                                                                         - - TATTAAATTT AGTAATAGCA AATGTTTATA GGATATTCAA ATTTCATTTG  - #                1700                                                                         - - AATTTTTAAT TATTTTTGCT ACAGGTAATA TTCCTTTAAA ATACGTATAT  - #                1750                                                                         - - AACGTACAGA GAATAACAGA CAATATGATC TAAGTAAATG TCGAATCAAT  - #                1800                                                                         - - CATTAGTTGC CCAGGGAAAT TTAAACATTA TAGATCATTT TTAAATAATA  - #                1850                                                                         - - CACATAGTTT TAATTTTTAC TGTGTGTATA GATGCATGAT TAAATGACTT  - #                1900                                                                         - - AAATATTAAA AAAAAAAAAA AA           - #                  - #                   1922                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2160 base - #pairs                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -       (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO:8:                          - - CGGCGACCTG TGGCGTGAGC GAGCACAGCC GGAACCCTCC ACTAGCTGGC  - #                  50                                                                         - - ATTCCTACCA TCCTTTATAG TGATGCTACA GGACAAAGAG GAATGGATAA  - #                 100                                                                         - - AAACATTGGT GAGCAACTCA ATAGAGCATA TGAAGCCTTC CGACAGGCAT  - #                 150                                                                         - - GCATGGATAG AGATTCAGCA GTAAGAGAGC TACAGCAAAA GACTGAGAAC  - #                 200                                                                         - - TATGAACAAA GAATACGCGA GCAACAGGAA CAGCTGTCAT TTCAACAAAA  - #                 250                                                                         - - CCTAATTGAC AGGCTGAAAT CACAGCTACT TCTCGTGGAT TCTAGTCGAG  - #                 300                                                                         - - ATAACAGTTA TGGCTATGTA CCTTTGCTTG AAGACAGTGA CAGAAGGAAG  - #                 350                                                                         - - AATAATTTGA CCCTTGATGA ACCACATGAT AAAGTGAAAC TAGGAACACT  - #                 400                                                                         - - GAGAGATAAG CAATCAAAGG TGAGACGACA AGAAGTTTCT TCTGGAAAAG  - #                 450                                                                         - - AATCCGCCAA GGGTCTCAAC ATCCCTCTGC ATCACGAAAG GGATAATATA  - #                 500                                                                         - - GAGAAGACTT TCTGGGACCT TAAAGAAGAA TTTCATAGGA TTTGCTTGCT  - #                 550                                                                         - - AGCAAAAGCA CAGAAAGATC ACTTAAGCAA ACTTAATATA CCAGATATTG  - #                 600                                                                         - - CAACTGACAC ACAGTGTTCT GTGCCTATAC AGTGTACTGA TAAAACAGAG  - #                 650                                                                         - - AAACAAGAAG CGCTGTTTAA GCCCCAGGCT AAAGATGATA TAAATAGAGG  - #                 700                                                                         - - TATGTCGTGC GTCACAGCTG TCACACCAAG AGGACTGGGC CGGGATGAGG  - #                 750                                                                         - - AAGATACCTC TTTTGAATCA CTTTCTAAAT TCAATGTCAA GTTTCCGCCT  - #                 800                                                                         - - ATGGACAATG ACTCTATTTT TCTACATAGC ACTCCAGAGG CCCCGAGCAT  - #                 850                                                                         - - CCTTGCTCCT GCCACACCTG AGACAGTGTG CCAGGACCGA TTTAATATGG  - #                 900                                                                         - - AAGTCAGAGA CAACCCAGGA AACTTTGTTA AAACAGAAGA AACTTTATTT  - #                 950                                                                         - - GAAATTCAGG GAATTGACCC CATAACTTCA GCTATACAAA ACCTTAAAAC  - #                1000                                                                         - - AACTGACAAA ACAAACCCCT CAAATCTTAG AGCGACGTGT TTGCCAGCTG  - #                1050                                                                         - - GAGACCACAA TGTGTTCTAT GTAAATACGT TCCCACTTCA AGACCCGCCT  - #                1100                                                                         - - GACGCACCTT TTCCCTCACT GGATTCCCCA GGAAAGGCTG TCCGAGGACC  - #                1150                                                                         - - ACAGCAGGTA ACTGTTTTGC ATTAACAAAT ATTTTATTAT GTGTGAACAC  - #                1200                                                                         - - ACATTTTATC ATACATGTAC AGATACAAAT CTGTTTTAAG TTATCAGGCA  - #                1250                                                                         - - TCCATTTAAA ATTAATGACT ATCCAGAGTT GAGGCTTTCA ATAAAATATG  - #                1300                                                                         - - TAAGTTCTGT ATTCAAGGAC ATGAATTTTG AATGTGACTG CGCTAAAGCT  - #                1350                                                                         - - TCCTTGTGAT ACTGTGGCGT GGCTTTCCCT GCTTCGTCCT CTTCAAGCAC  - #                1400                                                                         - - AGCTTGTTGA CATCAGTGCT CTAATGGATG CTTTATTAAA GTCAGTTACA  - #                1450                                                                         - - GGCAGTAAAT AATTTTTTTA AAACTTGTGT AGGTACACAT AATAATGTGT  - #                1500                                                                         - - AATTTTCCAT AAGTAGATAA TTGCACCAAA TATTCAAAAT AAACTGTCAT  - #                1550                                                                         - - TCAGCCTACT TGTGTTACAT TTCTAGTTAC AGCAGTACAG AGGTCTGTAG  - #                1600                                                                         - - TGTTTGGTTT GTTTACTAAC CTGACACTAA GCAGATATCC TTATACAGTT  - #                1650                                                                         - - TTCAAATAAT CCCTGCACAT GAATACTGTA ATCAAATCTC TTCTTTACTG  - #                1700                                                                         - - TTTGTGAAGC ACAAAGACTT TATAGCCCAT GAATCTAATC CTACCATCCT  - #                1750                                                                         - - TTCTTCCAGA TTCAGGTTCT TTCACAGAAA TATTCCTTTT TGTTAGGAAG  - #                1800                                                                         - - AAAAAAAGTT TTGTTTAATT TCTGAAGGTA AATGCTAAGT GTAGAAATGT  - #                1850                                                                         - - TAAAATAAAT AGAAGCATCT CATTAGAACT TTCAAACATT TGATTTTCTA  - #                1900                                                                         - - TCAGATTAAA AAAAATACTT AATACCTTTG GTTTACGTAT TCCTATCAGT  - #                1950                                                                         - - TATAGGCTTT TTGAACAGCA TGGAAAGAAG CAATAGTGAA GCTGTAGGAT  - #                2000                                                                         - - GTCTTAGTAG TGGGCGTAAG TAGAGATTCT GACAAGTCTT AATTATTAAC  - #                2050                                                                         - - TCTCTTATGT TCCACCCTGT ACCTTATTTC ACTTTATGGT CTCAGCTATA  - #                2100                                                                         - - GTTGCTACCA AATGAAACAA TTAAACAATT TCATGTGTTG CGAAAAAAAA  - #                2150                                                                         - - AAAAAAAAAA 2160                                                         __________________________________________________________________________

We claim:
 1. An assay for identifying a molecule the signal transductionof which is mediated by the association of a TRAF with a cellularprotein, comprising (a) incubating a mixture comprising said TRAF and anI-TRAF with a candidate molecule, and (b) detecting the ability of saidcandidate molecule to release TRAF from a complex formed with I-TRAF. 2.The assay of claim 1 wherein said mixture further comprises saidcellular protein, and the ability of said candidate molecule to initiateTRAF-mediated signaling through said cellular protein is detected. 3.The assay of claim 2 wherein said TRAF comprises the TRAF domain of anative TRAF1, TRAF2 or TRAF3 polypeptide.
 4. The assay of claim 2wherein said TRAF, I-TRAF and cellular protein are human.
 5. The assayof claim 4 wherein said I-TRAF comprises the N-terminal portion of anative human I-TRAF polypeptide.
 6. The assay of claim 2 wherein saidcellular protein is a member of the TNF receptor superfamily.
 7. Theassay of claim 2 wherein said cellular protein is selected from thegroup consisting of TNF-R2, CD40 and CD30.